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Elena Martynova, Qing Xie, Mark Emerson, Paul Overbeek, Ales Cvekl; Transcriptional regulation and function of Gata3 in lens differentiation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1714.
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© ARVO (1962-2015); The Authors (2016-present)
The lens architecture requires a synchronized cell cycle exit of lens precursor cells at the posterior part of the embryonic lens vesicle. This process is comprised of extracellular signaling, cytoplasmic signal transduction cascade, and nuclear control of gene expression. Transcription factor Gata3 has very restricted expression profile at the posterior part of the lens vesicle and its depletion abrogates cell cycle exit of the primary lens fiber cells; however, it is unclear how expression of Gata3 is regulated in lens and which genes are downstream of Gata3. The current project will determine the role of Gata3 in the cell cycle exit-coupled differentiation and regulation of its expression by extracellular signaling during mouse lens development.
Lens-specific Gata3 enhancers were predicted by analysis of lens chromatin and tested by luciferase reporter assays. Selected promoter-enhancer constructs driving EGFP expression were tested in transgenic mice. Immunofluorescence analysis of frozen tissues was performed using anti-EGFP antibodies. The role of Gata3 in lens development was studied through conditional gene knockout approach using Pax6-cre. Structure of Gata3 mutant lens was evaluated using H&E staining of paraffin sections. Gata3 binding at Cdkn1b/p27 and Cdkn1c/p57 loci in lens was analyzed by qChIP.
We identified and characterized a novel Gata3 lens-specific distal enhancer that recapitulates endogenous Gata3 expression during mouse lens development. Initial analysis of this region demonstrates presence of multiple putative binding sites for TFs regulated by BMP and FGF signaling. Conditional knockout of Gata3 leads to disruption of normal lens architecture and cataract formation. Immunofluorescence analysis demonstrates that expression of p27 and p57 is compromised in Gata3 mutants lens. Importantly, Gata3 binding was detected at both the Cdkn1b/p27 and Cdkn1c/p57 loci.
Collectively, the present studies show that Gata3 plays important roles in regulation of cell cycle exit during mouse lens development through direct regulation of p27 and p57. Presence of multiple binding sites for BMP- and FGF-regulated TFs at the Gata3 lens-specific enhancer suggests a novel model major signaling pathways regulate cell cycle exit-coupled differentiation through regulation of Gata3. Ongoing experiments are aimed to identify TFs upstream of Gata3 and its additional downstream target genes.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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